Buffers for stabilizing antigens

ABSTRACT

The present invention is directed to an antigen diluent or buffer for antigens, in particular HCV recombinant antigens, comprising a reducing agent. The antigen diluent or buffer serves as a stabilizing buffer for the antigens. The present invention is also directed to antigen diluents or buffers for use in an automated immunoassay.

[0001] This application claims priority benefit under 35 U.S.C. §119 toapplication Ser. No. 60/059,703, hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

[0002] The present invention is related generally to the field ofimmunoassays and specifically to buffers for stabilizing antigens, inparticular hepatitis C virus (HCV) antigens, for use in anti-HCVimmunoassays.

BACKGROUND OF THE INVENTION

[0003] In general, immunoassays are produced by first determiningepitopes that are specifically associated with a virus and thendetermining which of the epitopes is preferred for the assay beingdeveloped. When the particular epitopes are isolated, their sequencesare determined, and genetic material for producing the epitopes isproduced. Methods of producing proteins by either chemical or biologicalmeans are known, as are assays used to detect the presence of antibodiesto particular epitopes. Highly selective and sensitive immunoassaysgenerally contain major immunodominant epitopes of the pathogensuspected of infecting a patient.

[0004] For the virus HCV, major immunodominant linear epitopes have beenidentified from the core, NS3 (nonstructural), NS4 and NS5 regions ofthe virus polyprotein. HCV core protein and putative matrix proteinshave been assayed against human serum samples containing antibodies toHCV and several immunodominant regions within the HCV proteins have beendefined. Sallberg, et al., J. Clin. Microbiol., 1992, 30, 1989-1994,incorporated by reference herein in its entirety. Protein domains ofHCV-1 polyproteins including domains C, E1, E2/NS1, NS2, NS3, NS4, andNS5 have been identified and their approximate boundaries have beenprovided in WO 93/00365, incorporated by reference herein in itsentirety. In addition, individual polypeptides having sequences derivedfrom the structural region of HCV have been designed in order to obtainan immunodominant epitope useful in testing sera of HCV patients.Kotwal, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 4486-4489,incorporated by reference herein in its entirety.

[0005] The current assay of choice for HCV antibody detection is theOrtho 3.0 ELISA, a manual assay. Chiron-produced recombinant HCVantigens for use in the ELISA are c200 (ns-3, c100), c22 and NS-5. Thec33c and c22 antigens are very immunogenic. Antibodies to c33c and c22are also found in early seroconversion panels. The prevalence of HCVantibodies varies from 58% to 95% with the highest detection rateobtained for the c33c polypeptide followed by the c22 polypeptide.Chien. et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10011-10015,incorporated by reference herein in its entirety. However, problems ofstabilizing HCV antigens in the liquid phase have been encountered. Thelack of stability of HCV antigens in the liquid phase is a majordisadvantage of the current HCV antibody detection assay. Therefore,developing an antigen buffer for the anti-HCV immunoassay has beenattempted utilizing the same antigens as the Ortho 3.0 ELISA wherein thebuffer stabilizes the HCV antigens. In addition, adapting the reagents,buffer and protocols to already existing automated machines, such as theACS:Centaur has been attempted. Accordingly, there is currently a needto improve the stability of HCV antigens in the liquid phase for use inanti-HCV immunoassays. Such improved assay reagents and methods providefor better detection of HCV antibodies in screening of blood suppliesand other biological fluids. It is contemplated that the buffers be canused for other antigens which may be unstable in the liquid phase, e.g.human immunodeficiency virus (HIV) antigens.

SUMMARY OF THE INVENTION

[0006] In one aspect, the present invention is directed to an antigendiluent or buffer capable of stabilizing antigens in the liquid phase,in particular HCV recombinant antigens, comprising a reducing agent.

[0007] In another aspect, the present invention is directed toimmunoassays using an antigen diluent or buffer containing a reducingagent.

[0008] In another aspect, an improved immunoassay kit is provided, theimprovement comprising using an antigen diluent or buffer for HCVantigens containing a reducing agent.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of virology, immunology,microbiology, molecular biology and recombinant DNA techniques withinthe skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); DNA Cloning: A Practical Approach, Vols. I &II (D. Glover, ed.); Methods In Enzymology (S. Colowick and N. Kaplaneds., Academic Press, Inc.); Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell ScientificPublications); and Fundamental Virology, 2nd Edition, Vols. I & II (B.N. Fields and D. M. Knipe, eds.).

[0010] Reagent stability over time is a critical issue. The c33c antigendiluted in buffer and tested the same day was functional using MagicLite Assay protocols described below. However the reagent, when stressedat 37° C., lost more than 50% immunoreactivity to early seroconversionpanels. The c33c in the liquid phase may slowly “aggregate” or becomeinsoluble. Known components were tried in order to stabilize c33cimmunoreactivity such as sugars, gelatin, glycerol, cross-linkingreagents and anti-oxidants. It was discovered that keeping the c33cantigen in the reduced form can maintain immunoreactivity for periodsover 24 hours, even up to at least 7 days, at 37° C. on early c33cseroconversion panels (matching Ortho 3.0 ELISA performance). Thereducing agent reduces the disulfide bonds among cysteine groups withinthe c33c molecule, perhaps improving c33c immunoreactivity andsolubility. There was no indication of antigen stability at 37° C. C forsuch lengths of time of conventional lite reagents in the liquid phaseprior to the advent of the antigen diluent for c33c. Similar experimentswere performed for c200 and a multiple epitope fusion antigen (MEFA-6)as shown below. Thus, the present invention provides antigen diluents orbuffers for stabilizing HCV antigens for use in anti-HCV immunoassays.The antigen diluents or buffers of the present invention can be used inimmunoassays such as, for example, ELISA and CLIA.

[0011] The present invention is directed to antigen diluents or buffersproviding for improved stability of HCV antigens in the liquid phase. Asused herein, “antigen diluents or buffers” refers to the solution inwhich the antigen is contained; it may or may not possess bufferingcapacity. In particular, the invention is directed to antigen diluentsor buffers for improved stability for the recombinant HCV antigens inthe Ortho 3.0 ELISA, and the like. The present invention was achieved byadding a reducing agent such as, for example, dithiothreitol (DTT) tothe antigen diluent or buffer.

[0012] In a preferred embodiment of the invention, the HCV antigendiluent or buffer comprises a reducing agent. In another preferredembodiment of the invention, the HCV antigen diluent or buffer comprisessodium phosphate (pH 6.5), ethylenediaminetetraacetic acid (EDTA), DTT,gelatin, ammonium thiocyanate, sodium azide and SDS. However, theseindividual reagents can be replaced by similar reagents performingessentially the same function. For example, DTT can be replaced withadditional reducing agents such as, for example, thioglycerol,mercaptoethenol, and the like. Sodium phosphate can be replaced bysodium borate and other buffers. Gelatin can be replaced with BSA andother blocking agents of non-specific binding. Sodium thiocyanate can bereplaced with ammonium thiocyanate and other chaotropic agents. SDS canbe replaced by a number of detergents such as, for example, Tween-20,and other detergents. Sodium azide can be replaced by otheranti-bacterial agents. In addition, EDTA can be replaced by ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) andother chelating agents. One skilled in the art is familiar with reagentswhich can be substituted for those of the present invention.

[0013] In a preferred embodiment of the present invention, the HCVantigen diluent comprises from about 15 mM to about 100 mM sodiumphosphate, pH 6.5. More preferably the diluent comprises from about 20mM to about 75 mM sodium phosphate, pH 6.5. Most preferably, the diluentcomprises 24 or 25 mM sodium phosphate, pH 6.5.

[0014] In another preferred embodiment of the present invention, the HCVantigen diluent comprises from about 1 mM to about 10 mM EDTA. Morepreferably the diluent comprises from about 3 mM to about 7 mM EDTA.Most preferably, the diluent comprises 5 mM EDTA.

[0015] In another preferred embodiment of the present invention, the HCVantigen diluent comprises from about 1 mM to about 200 mM DTT. Morepreferably the diluent comprises from about 5 mM to about 100 mM DTT.Most preferably, the diluent comprises 10 mM DTT.

[0016] In another preferred embodiment of the present invention, the HCVantigen diluent comprises from about 0.05% to about 1% gelatin. Morepreferably the diluent comprises from about 0.1% to about 0.5% gelatin.Most preferably, the diluent comprises 0.2% gelatin.

[0017] In another preferred embodiment of the present invention, the HCVantigen diluent comprises from about 10 mM to about 500 mM ammoniumthiocyanate. More preferably the diluent comprises from about 50 mM toabout 200 mM ammonium thiocyanate. Most preferably, the diluentcomprises 100 mM ammonium thiocyanate.

[0018] In another preferred embodiment of the present invention, the HCVantigen diluent comprises from about 0.01% to about 0.3% sodium azide.More preferably the diluent comprises from about 0.05% to about 0.2%sodium azide. Most preferably, the diluent comprises 0.09% sodium azide.

[0019] In another preferred embodiment of the present invention, the HCVantigen diluent comprises from about 0.01% to about 0.5% SDS. Morepreferably the diluent comprises from about 0.05% to about 0.2% SDS.Most preferably, the diluent comprises 0.1% SDS.

[0020] In another preferred embodiment of the present invention, the HCVantigen diluent for the manual assay comprises 25 mM sodium phosphate,pH 6.5, 5 mM EDTA, 10 mM DTT, 0.2% gelatin, 100 mM ammonium thiocyanate,0.09% sodium azide and 0.1% SDS.

[0021] For the automated assays, a preferred antigen buffer for c33ccomprises 50 mM phosphate, 5 mM EDTA, 100 mM ammonium thiocyanate, 0.06%SDS, 0.25% fish gelatin and 10 mM DTT.

[0022] Table 1 shows a preferred HCV buffer. TABLE 1 HCV Antigen BufferFor HCV Antigens Description Concentration Source Product # Lot # Sodium25 mM JT Baker 3818-05 A45101 Phosphate Monobasic Sodium 0.09% FisherBP922-500 953331 Azide Biotech EDTA 5 mM Fisher S311-100 953493 ChemicalSodium 100 mM Sigma S-7757 96HO543 Thiocyanate Tween-20 0.10% SigmaP-1379 56HO876 Gelatin (fish) 0.20% Sigma G-7765 45H1157 DTT 10 mM SigmaD-5545 26HO3801

[0023] The HCV antigen diluents or buffers of the present invention canbe prepared by well known media preparation techniques. A preferredembodiment of preparing the HCV antigen diluents of the presentinvention is shown in Table 2. TABLE 2 Process For Preparation OfDiluents Process Step Amount 1. Add 95% of batch quantity P-30 water 2.Add sodium phosphate, monobasic 3.45 g/L 3. Add sodium azide 0.9 g/L 4.Add EDTA 1.86 g/L 5. Add sodium thiocyanate 8.1 g/L 6. pH solution 6.5 ±0.1 and stir 7. Add Tween-20 1 mL/L 8. Add gelatin 2 mL/L 9. Add DTT1.54 g/L 10. Stir solution until dissolved 11. Filter through 1.22 μmMillipak filter unit 12. Store at 4° C. in dark

[0024] The HCV antigen diluents or buffers of the present invention canbe used in manual or automatic assays. The antigen diluents or buffersof the present invention can be used with numerous HCV antigensincluding, but not limited to, c33c, MEFA-6, c22p, c100p, NS-5 and c200.These HCV antigens can be prepared by recombinant procedures routinelyused in the art.

[0025] HCV c33c (NS3) and c100 (NS4) region sequences contain epitopesfrom the immunodominant core and were prepared as described in Chien, etal., Proc. Natl. Acad. Sci. USA, 1992, 89, 10011-10015. The c200 antigenis a fusion protein consisting of the c33c and c100 antigens. The c22(119 amino acids) and NS5 (942 amino acids) antigens were expressed asinternal antigens within the yeast S. cerevisiae as C-terminal fusionswith human superoxide dismutase (SOD) using methods described previouslyfor the generation of the c100-3 (363 amino acids) antigen. Kuo, et al.,Science, 1989, 244, 362-364, incorporated herein by reference in itsentirety; and Cousens, et al., Gene, 1987, 61, 265-275, incorporatedherein by reference in its entirety. The c33c antigen (363 amino acids)was expressed as an internal SOD fusion polypeptide in E. coli bymethods described for the synthesis of 5-1-1 antigen. Choo, et al.,Science, 1989, 244, 359-362, incorporated herein by reference in itsentirety. The recombinant HCV antigens were purified as described inChien, et al., Proc. Natl. Acad. Sci. USA, 1989, 89, 10011-10015. In thepresent invention, all HCV antigens were prepared as SOD fusionproteins. However, other suitable fusion proteins can be made dependingupon the availability of appropriate antibodies that recognize thefusion partner.

[0026] MEFA-6 contains epitopes from the core, envelope, NS3, NS4 andNS5 regions of the hepatitis C polyprotein, including equivalentantigenic determinants from HCV strains 1, 2, and 3. The various DNAsegments coding for the HCV epitopes were constructed by PCRamplification or by synthetic oligonucleotides. Table 3, below,describes the amino acid segments of each epitope, the lineararrangement of the various epitopes and the number of copies in theMEFA-6 cassette. MEFA-6 cassette was prepared as described inapplication PCT U.S. Ser. No. 97/08950 filed May 23, 1997, incorporatedherein by reference in its entirety.

[0027] As shown in Table 3, the MEFA-6 antigen includes multiple copiesof HCV epitopes from the core and NS5 region; different serotypeepitopes from the NS4 5-1-1 region; a single copy of major linearepitopes from the c100 C-terminal regions, E1, and E2 regions, as wellas the HCV NS3 (c33c) region. The general structural formula for MEFA-6is hSOD—E1-E2-c33c-5-1-1 (type 1)-5-1-1 (type 3)-5-1-1 (type2)-c100-NS5(2 copies)-core (2 copies). This antigen has a very highexpression level in yeast, purifies to a high degree of homogeneity, andexhibits high sensitivity and high selectivity in the immunoassaysdescribed below. MEFA-6 was prepared as described in application Ser.No. 08/859,524 filed May 20, 1997, incorporated herein by reference inits entirety. TABLE 3 MEFA-6 Antigen Epitopes And Their Location WithinThe HCV Genome 5′ End MEFA aa# Site Epitope HCV aa# Strain 1-154 NcoIhSOD 159-176 EcoRI E1 303-320 1 179-217 HindIII E2 405-444 1 218-484DraIII c33c 1192-1457 1 487-533 SphI 5-1-1 1689-1735 1 536-582 NruI5-1-1 1689-1735 3 585-631 ClaI 5-1-1 1689-1735 2 634-673 AvaI c1001901-1940 1 676-711 XbaI NS5 2278-2313 1 714-749 BglII NS5 2278-2313 1750-793 NcoI core 10-53 1 796-839 Sad core 10-53 1

[0028] The detectable marker may include, but is not limited to, achromophore, an antibody, an antigen, an enzyme, an enzyme reactivecompound whose cleavage product is detectable, rhodamine or rhodaminederivative, biotin, streptavidin, a fluorescent compound, achemiluminescent compound, derivatives and/or combinations of thesemarkers. In the present examples, the chemiluminescent compound dimethylacridinium ester (DMAE, Ciba Corning Diagnostics Corp.) was used.Labeling with any marker is carried out under conditions for obtainingoptimal detection and antigenicity of the MEFA-6 or other epitope. WhereDMAE is the detectable marker in an assay, the resultant HCV r-Ag-DMAEconjugate is the tracer, with DMAE detectable by light emission whenreacted with NaOH/H₂O₂.

[0029] A polypeptide, antibody or synthetic peptide antigen was labeledwith DMAE by reaction of amino acid side chains (e.g. lysine ε sidechain or cysteine thiol) with a reactive moiety covalently linked toDMAE (see WO 95/27702, published Oct. 19, 1995, Ciba Corning DiagnosticsCorp., herein incorporated by reference in its entirety). For example,the HCV antigens described herein were labeled by reaction with theamino groups of lysine side chains with NSP-DMAE-NHS(2′,6′-Dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl-10-(3′-Sulfopropyl)-acridinium-9-carboxylate)obtained from Ciba Corning. Thiols of amino acid side chains can belabeled using DMAE-ED-MCC or NSP-DMAE-PEG-BrAc (Ciba Corning). Labelingprocedures were generally as described in WO 95/27702 with variations inconditions as necessary for each antigen to provide optimal detectionand antigenicity.

EXAMPLES Example 1 Manual Assay

[0030] A Magic Lite Analyzer System II (MLA II) is used for the manualassay. Parameters such as volume, concentration, time, and temperatureare provided for guidance, but may be adjusted accordingly. Briefly, a10 μl aliquot of test sample was added to corresponding tubes. The testsample is preferably a biological fluid (plasma or serum, for example)possibly containing anti-HCV antibodies, as well as proper controls. Toeach tube is added 100 μl of antigen diluent or buffer and incubated for6 minutes at 37° C. To each tube is added 100 μl of solid phase buffercontaining paramagnetic particles (PMP) conjugated to rat anti-human IgGantibodies (PMP/anti-human IgG) for a final concentration ofapproximately 60 μg/assay. However, other anti-human IgG antibodies aresuitable. Preferably, the paramagnetic particles are less thanapproximately 10 μm in diameter. The PMP/anti-human IgG particles can bediluted in a diluent containing Tris buffer, pH 8.0, 150 mM NaCl, 2.75%BSA, 0.1% casein, 0.1% Tween-20, 0.1% yeast extract, 0.25% E. coliextract, 0.005% SOD, 0.09% NaN₃ and 1 mM EDTA. Subsequently, recombinantHCV antigens (HCV antigen/SOD fusion proteins) conjugated to DMAE(MEFA-6-DMAE, c33c-DMAE and c200-DMAE, for example) are added in a 50 μlvolume of ligand reagent (LR) diluent at a concentration ofapproximately 0.1 μg/assay to 1 μg/assay. Preferably, an amount ofligand reagent is added to each sample such that approximately 25×10⁶light unit equivalents (relative light units, RLU) are present perassay. This approximate amount of light unit equivalents is preferredfor the addition of a single ligand, or for multiple ligands. LR diluentcontains Tris buffer, pH 8.0, 150 mM NaCl, 1.0% BSA, 0.1% Tween-20,0.09% NaN₃, and 1 mM EDTA. To ensure complete mixing, the tubes areshaken on a Vortex mixer 6 times at 5-10 seconds each time. The sampletubes are incubated at 37° C. for 18 minutes. The sample tubes areplaced on a magnet for 3 minutes, for sufficient time to sediment thePMP particles. The samples are decanted using a magnet to retain the PMPparticles. The PMP particles are washed twice with vortexing in 1 ml ofPBS. The wash solution is PBS, 0.1% Tween-20, 0.09% NaN₃, and 1 mM EDTA.The steps of mixing, incubating, sedimenting and decanting may berepeated at least one time. To each tube 100 μl of water is added toresuspend the PMP particles. The tubes are then placed in an MLA-IIinstrument and light emission is measured for 2 seconds.

Example 2 Automated Assay

[0031] The manual anti-HCV assay described above was adapted forautomated use using an ACS:Centaur apparatus. The following procedure isused. Briefly, the ACS:Centaur system automatically performs thefollowing steps: 1) dispenses 10 μl of sample into a cuvette; 2)dispenses 100 μl of ancillary diluent buffer, 100 μl of LiteReagent/Solid Phase, 50 μl of antigen reagent 2 (e.g., MEFA-6), 50 μl ofantigen reagent 1 (e.g., c33c) and incubates the mixture for 18 minutesat 37° C.; 3) separates the solid phase from the mixture and aspiratesthe unbound reagent; 4) washes the cuvette with wash reagent 1; 5)dispenses 300 μl each of acid reagent and base reagent to initiate thechemiluminescent reaction; and 6) reports results according to theselected option, as described in the system operating instructions or inthe online help system. The solid phase/Lite reagent diluent buffercomprises 50 mM Tris, 0.5 M KCl, 1 mM EDTA, 3.75% BSA, 0.003% Yeast,0.05 g/L E. coli, 0.5% Tween-20, 2 mg/L Amphotericin B, 24 mg/LGentamicin Sulfate, 30 μg/test Solid Phase and 45×10⁶ test Lite Reagent(anti-SOD*DMAE antibodies). The ancillary diluent buffer comprises 50 mMTris, 0.5M KCl, 1 mM EDTA, 3.75% BSA, 0.003% Yeast, 0.05 g/L E. coli,0.5% Tween-20, 2 mg/L Amphotericin B, 24 mg/L Gentamicin Sulfate, 0.05g/L Ascites IgG1 and 0.1 g/L Ascites 1gG2A (blocking antibodies). Thewash reagent comprises PBS/Tween-20. The acid reagent comprises 0.5%H₂O₂/0.1 N HNO₃. The base reagent comprises <0.25N NaOH with surfactant.

Example 3 Manual Assay with c33c

[0032] A manual assay using c33c HCV antigen was performed with 100 ngof c33c per assay using the methodology described above in Example 1.The antigen diluent comprised 25 mM sodium phosphate, pH 6.5, 100 mMsodium thiocyanate, 5 mM EDTA, 0.1% Tween-20, 0.2% fish gelatin, 0.09%sodium azide and 10 mM DTT. The assay was performed with 3×10⁶ RLU/10μl, 30 μg/assay PMP. The assay was performed at varying times and undervarying temperatures. For example, the assay was performed at Day 0 at4° C., at Day 3 at 4° C., at Day 1 at 37° C., at Day 2 at 37° C., at Day3 at 37° C. and at Day 6 at 37° C.

[0033] A 10 μl sample (such as a biological fluid containing humananti-HCV antibodies) was added to each sample tube. Samples included:random negative controls (r1, r2 and r3), a positive control (Virotrol),seroconversion panels (PHV905-5, PHV907-4 and PHV904-6), HCV patientsamples (FF25931) and seroconversion samples (6214-09 and 6212-04). Theresults are shown in Table 4. Sensitivity was reported as the opticaldensity of the assay sample divided by the assay detection cut off inoptical density units (s/co). All known negative samples exhibitedrelative light units (RLU) below the cutoff value, while known positivesamples exhibited RLUs well above the cutoff value.

[0034] For comparative purposes, the detection of HCV antibodies fromsome of the samples (see Table 4) was also performed by Ortho 3.0 and acommercial strip immunoblot assay (RIBA® 3.0 Chiron Corporation), whichassay is used clinically as a confirmatory test for HCV antibodydetection. According to the RIBA® method, recombinant HCV antigens areseparated by gel electrophoresis and contacted with patient serum.Reactivity with the separated antigens is performed by immunoblot assayusing secondary labeled antibodies. Assay results are scored on aplus/minus scale. Eheling, et al., Lancet, 1991, 337, 912-913,incorporated herein by reference in its entirety. The Ortho 3.0 assaywas performed according to the manufacturer's instructions. c33c, c22p,c100p, and NS-5 were used as the HCV antigens for these tests.

[0035] Briefly, the RIBA® 3.0 assay was performed as follows.Approximately 30 minutes before beginning the assay, the kit was removedfrom refrigeration (2 to 8° C.) and the components of the kit allowed tocome to room temperature (15 to 30° C.). The required number of stripswere removed from the sealed foil pouches and placed in the assay tuberack in their respective tubes. One ml of Specimen Diluent was added toeach tube so that the entire strip was covered with liquid. Twenty μl ofthe appropriate specimen or control was added to the corresponding tube.The tubes were capped and inverted to mix. The rack with the tubes wasplaced on a rocker and fastened with rubber bands or tape; the rack wasrocked (at 16-20 cycles/minute) for 4 to 4% hours at room temperature(15 to 30° C.). The tubes were uncapped and the liquid was completelyaspirated into a waste container. One ml of Specimen Diluent was addedto each tube. The tubes were capped and placed on the rack on the rockerand rocked for 30 to 35 minutes at room temperature. The liquid was thenaspirated. One ml of Working Wash Buffer was added to each tube, thenthe liquid and strips poured into wash vessels containing 30 ml ofWorking Wash Buffer (maximum 20 strips per wash vessel). The washvessels were completely filled with Working Wash Buffer (60 mL totalvolume), then the wash was decanted. To retain the strips, the washvessel was gently rolled while decanting. Sixty ml of Working WashBuffer was added, swirled, then the wash was decanted the whileretaining the strips. One ml of Conjugate per strip was added to eachwash vessel (minimum 10 ml per wash vessel). The wash vessels wererotated on a rotary shaker at 110±5 rpm for 9 to 11 minutes at roomtemperature (15 to 30° C.). Working Substrate was prepared up to 1 hourprior to use. Upon completion of Conjugate incubation, the Conjugate wasdecanted and the strips were washed by adding 60 ml of Working WashBuffer and swirling. The wash was decanted and this step was repeatedtwo more times. The final wash was decanted. One ml of Working Substratewas added per strip to each wash vessel (minimum 10 ml per wash vessel).The wash vessels were rotated on a rotary shaker at 110±5 rpm for 15 to20 minutes at room temperature (15 to 30° C.). The Working Substrate wasdecanted and the strips were washed by adding 60 ml of distilled ordeionized water and swirling. The wash was decanted and this step wasrepeated one more time. To retain strips, the wash vessel was gentlyrolled while decanting. Using forceps, the strips were transferred toabsorbent paper and excess water was blotted. The strips were air-driedin the dark for at least 30 minutes at room temperature. The strips wereinterpreted within 3 hours. Anti-HCV reactivity in a specimen wasdetermined by comparing the intensity of each antigen band to theintensity of the human IgG (Level 1 and Level II) internal control bandson each strip. The identity of the antibodies was defined by thespecified location of the antigen band. The intensity of theantigen/peptide bands was scored in relation to the intensities of theinternal IgG controls as follows: absent (−), less than intensity of theLevel I IgG control band (−/+), equal to intensity of the Level I IgGcontrol band (1+), greater than intensity of the Level I IgG controlband and less than intensity of the Level II IgG control band (2+),equal to intensity of the Level II IgG control band (3+), and greaterthan intensity of the Level II IgG control band (4+).

Example 4 Manual Assay with c200

[0036] A manual assay using c200 HCV antigen was performed as describedin Example 1 with various amounts of reducing agent. The stabilizingbuffer was the same as in Example 3, except for the amount of reducingagent. The assay was performed with 3×10⁶ RLU/10 μl, 30 μg/assay PMP.The assay was performed at varying times and under varying amounts ofreducing agent. For example, the assay was performed after 1 day at 37°C. with 20 mM DTT (Vial I), after 1 day at 37° C. without DTT (Vial II),and after 1 day at 37° C. where 20 mM DTT was added prior to testing(Vial III). Vials II and III were also tested after 3 days.

[0037] A 10 μl sample (such as a biological fluid containing humananti-HCV antibodies) was added to each sample tube. Samples included:random negative controls (r1, r2, r3, r4 and r5), seroconversion panels(PHV904-6 and PHV906-1) and HCV patient samples (FF25931) at variousdilutions. The results are shown in Table 5. s/n is the sensitivitydivided by the value ave.neg.

Example 5 Manual Assay with MEFA-6 and c33c

[0038] A manual assay using MEFA-6 and c33c HCV antigen was performedwith 100 ng of MEFA-6 and 85 ng of c33c per assay using the methodologydescribed above in Example 1. The stabilizing buffer for MEFA-6comprised 50 mM sodium borate, pH 9.5, 5 mM EDTA, 0.05% Tween-20, 0.5%BSA, and 1% thioglycerol. At this pH 9.5 MEFA-6 is stable so no reducingagent is necessary. The buffer for c33c comprised 25 mM sodiumphosphate, pH 6.5, 5 mM EDTA, 0.1% Tween-20, 0.2% fish gelatin, 100 mMsodium thiocyanate, and 10 mM DTT. The assay was performed with 4.5×10⁶RLU/10 μl of anti-SOD*DMAE, 30 μg/assay PMP. The assay was performed atvarying times and under varying temperatures. For example, the assay wasperformed at Day 7 at 4° C. and at Day 7 at 37° C.

[0039] A 10 μl sample (such as a biological fluid containing humananti-HCV antibodies) was added to each sample tube. Samples included,random negative controls (r1, r2, r3 and r4), a positive control(Virotrol), seroconversion panels (PHV905-5, PHV909-1, PHV909-2 andPHV909-3), seroconversion samples (6212-02 and 6214-09) andseroconversion control panels (SC-0030A, SC-0030B, SC-0030C, SC-0030D,SC-0040A, SC-0040B, SC-0040C, SC-0040D and SC-0040E). The results areshown in Table 6. Sensitivity was reported as the optical density of theassay sample divided by the assay detection cut off in optical densityunits (s/co). All known negative samples exhibited relative light units(RLU) below the cutoff value, while known positive samples exhibitedRLUs well above the cutoff value.

[0040] For comparative purposes, the detection of HCV antibodies fromsome of the samples (see Table 6) was also performed by Ortho 3.0 andRIBA® 3.0 as described in Example 3.

Example 6 Manual Assay with MEFA-6

[0041] A manual assay using MEFA-6 HCV antigen was performed with 100 ngof MEFA-6 per assay using the methodology described above in Example 1.The buffer for MEFA-6 comprised 50 mM sodium borate, pH 9.5, 5 mM EDTA,0.05% Tween-20, 0.5% BSA, and 1% thioglycerol. The assay was performedwith 4.5×10⁶ RLU/10 μl of anti-SOD*DMAE, 30 μg/assay PMP. The assay wasperformed at Day 7 at 4° C.

[0042] A 10 μl sample (such as a biological fluid containing humananti-HCV antibodies) was added to each sample tube. Samples included,random negative controls (r1, r2 and r3), positive control (Virotrol)and seroconversion control panels (SC-0030A, SC-0030B, SC-0030C andSC-0030D). The results are shown in Table 7. Sensitivity was reported asthe optical density of the assay sample divided by the assay detectioncut off in optical density units (s/co). All known negative samplesexhibited relative light units (RLU) below the cutoff value, while knownpositive samples exhibited RLUs well above the cutoff value.

[0043] For comparative purposes, the detection of HCV antibodies fromsome of the samples (see Table 7) was also performed by Ortho 3.0 andRIBA® 3.0 as described above.

[0044] The foregoing examples are meant to illustrate the invention andare not to be construed to limit the invention in any way. Those skilledin the art will recognize modifications that are within the spirit andscope of the invention. All references cited herein are herebyincorporated by reference in their entirety. TABLE 4 c33c Assay Day 0Day 3 Day 1 Day 2 Day 3 Day 6 Sample 4° C. 4° C. 37° C. 37° C. 37° C.37° C. r1 1925 1247 1001 1509 1971 2202 r2 1740 1679 1632 1448 1401 1863r3 1602 1432 1463 1401 1725 1940 Virotrol 61708 64156 65604 60060 5659559044 6214-09 14322 16555 10888 16555 14784 15246 6212-04 40856 4335141842 40225 36421 39008 PHV905-5 10734 15030 14969 13721 15785 13999PHV907-4 2341 1756 1940 2017 2110 2002 P2HV904-6 53299 49496 54208 5428547155 45676 FF25931 1:8 567120 608993 530006 572603 568974 581504 ave.neg. 1756 1453 1365 1453 1699 2002 cutoff 5267 4358 4096 4358 5097 6005ORTHO s/co s/co s/co s/co s/co s/co RIBA 3.0 3.0 Virotrol 11.72 14.7216.02 13.78 11.10 9.83 c33c c22p c100p NS-5 s/co 6214-09 2.72 3.80 2.663.80 2.90 2.54 2+ − +/− − 0.9 6212-04 7.76 9.95 10.22 9.23 7.15 6.50 1+− − − 1.4 PHV905-5 2.04 3.45 3.65 3.15 3.1 2.33 1+ − − − 0.9 PHV907-40.44 0.40 0.47 0.46 0.41 0.33 − 1+ − − 0.1 PHV904-6 10.12 11.36 13.2312.46 9.25 7.61 2+ − − − >5.0

[0045] TABLE 5 c200 Assay Vial I Vial II Vial III Vial III Vial III VialII 37° C. Day 1 37° C. Day 1 37° C. Day 1 37° C. Day 1 37° C. Day 3 37°C. Day 3 control c200 test c200 test c200 test c200 test c200 test c200Sample 20 mM DTT w/o DTT added 20 mM DTT added 40 mM DTT added 40 mM DTTw/o DTT random r1 1463 1448 1078 1109 1217 801 random r2 1217 1324 12471879 1309 1016 random r3 1155 1247 1340 1217 1063 1124 random r4 11701217 2402 1340 1386 1140 random r5 1155 1232 1217 1494 serocon. PHV904-629106 10102 8763 13182 14060 2141 serocon. PHV906-1 27828 15231 1601621468 30523 25656 FF25931 1:4 643551 574944 435543 318025 322307 277616FF25931 1:256 26657 19774 16339 16524 22484 21699 FF25931 1:1024 99487854 8516 8408 12859 17048 ave.neg. 1232 1294 1457 1408 1244 1020 s/ns/n s/n s/n s/n s/n serocon. PHV904-6 23.6 7.8 6.0 9.4 11.3 2.1 serocon.PHV906-1 22.6 11.8 11.0 15.2 24.5 25.1 FF25931 1:4 522.4 444.5 299.0225.9 259.1 272.1 FF25931 1:256 21.6 15.3 11.2 11.7 18.1 21.3 FF259311:1024 8.1 6.1 5.8 6.0 10.3 16.7

[0046] TABLE 6 MEFA-6 + c33c Assay 40° C. Day 7 37° C. Day 7 Ortho 3.0RIBA 3.0 Sample s s/co s s/co s/co c100p c33c c22p NS-5 Genotype NABISC-0030A 6607 0.39 5960 0.43 0.005 − − − − 1a NABI SC-0030B 14522 0.868778 0.64 0.015 3+ +/− +/− − NABI SC-0030C 86748 5.12 46785 3.40 1.8374+ 1+ 2+ − NABI SC-0030D 472749 27.92 489304 35.54 4.900 4+ 4+ 4+ 3+NABI SC-0040A 9379 0.55 7454 0.54 0.003 − − − − 2b NABI SC-0040B 127200.75 7546 0.55 0.056 − − − − NABI SC-0040C 65927 3.89 29799 2.16 1.215+/− 2+ − − NABI SC-0040D 106845 6.31 43613 3.17 1.534 +/− 2+ − − NABISC-0040E 175067 10.34 78124 5.67 3.247 1+ 3+ 1+ − random r1 5236 4697random r2 5652 4112 random r3 5991 5375 random r4 5698 4173 Ortho 3.0RIBA 3.0 control Virotrol I 117548 6.94 74721 5.43 s/co c100p c33c c22pNS-5 BCP 6212-04 68807 4.06 31616 2.30 1.4 − 1+ − − BCP 6214-09 815434.82 24270 1.76 0.9 +/− 2+ − − BBI PHV905-5 25040 1.48 16755 1.22 0.9 −1+ − − BBI PHV909-1 6699 0.40 5313 0.39 0.0 − − − − BBI P5V909-2 306611.81 15646 1.14 1.3 − − 1+ +/− BBI PHV909-3 32432 1.92 16570 1.20 1.4 −− 2+ +/− ave. neg. 5644 4589 cutoff 16933 13768

[0047] TABLE 7 MEFA-6 Assay 4° C. Day 7 Sample s s/co random r1 8624random r2 8609 random r3 7192 Ortho 3.0 RIBA 3.0 control 129406  5.00s/co c100p c33c c22p NS-5 Genotype Virotrol I NABI SC-0030A 8516 0.330.005 − − − − 1a NABI SC-0030B 26827  1.04 0.015 3+ +/− +/− − NABISC-0030C 179980  6.96 1.837 4+ 1+ 2+ − NABI SC-0030D 508831  19.67 4.900 4+ 4+ 4+ 3+ ave. neg. 8624 cutoff 25872 

We claim:
 1. An antigen diluent or buffer comprising a reducing agent.2. An antigen diluent or buffer for Hepatitis C (HCV) antigenscomprising a reducing agent.
 3. The antigen diluent or buffer of claim 1or 2 wherein the reducing agent is selected from the group consisting ofdithiothreitol, thioglycerol and mercaptoethanol.
 4. The antigen diluentor buffer of claim 1 or 2 wherein the reducing agent is dithiothreitol(DTT).
 5. The antigen diluent or buffer of claim 4 wherein theconcentration of DTT is from about 1 mM to about 200 mM.
 6. The antigendiluent or buffer of claim 4 wherein the concentration of DTT is fromabout 5 mM to about 100 mM.
 7. The antigen diluent or buffer of claim 4wherein the concentration of DTT is about 10 mM.
 8. The antigen diluentor buffer of claim 1 or 2 further comprising a buffering agent.
 9. Theantigen diluent or buffer of claim 8 wherein the buffering agent isselected from the group consisting of sodium phosphate or sodium borate.10. The antigen diluent or buffer of claim 9 wherein the buffering agentis sodium phosphate.
 11. The antigen diluent or buffer of claim 10wherein the concentration of sodium phosphate, pH 6.5, is from about 15mM to about 100 mM.
 12. The antigen diluent or buffer of claim 1 or 2further comprising a detergent.
 13. The antigen diluent or buffer ofclaim 12 wherein the detergent is selected from the group consisting ofsodium dodecyl sulfate (SDS) and Tween-20®.
 14. The antigen diluent orbuffer of claim 13 wherein the detergent is SDS.
 15. The antigen diluentor buffer of claim 14 wherein the concentration of SDS is from about0.01% to about 0.5%.
 16. The antigen diluent or buffer of claim 1 or 2further comprising an anti-bacterial agent.
 17. The antigen diluent orbuffer of claim 16 wherein the anti-bacterial agent is sodium azide. 18.The antigen diluent or buffer of claim 17 wherein the concentration ofsodium azide is from about 0.01% to about 0.3%.
 19. The antigen diluentor buffer of claim 1 or 2 further comprising a chelating agent.
 20. Theantigen diluent or buffer of claim 19 wherein the chelating agent isethylenediaminetetraacetic acid (EDTA).
 21. The antigen diluent orbuffer of claim 20 wherein the concentration of EDTA is from about 1 mMto about 10 mM.
 22. The antigen diluent or buffer of claim 1 or 2further comprising a blocking agent of non-specific binding.
 23. Theantigen diluent or buffer of claim 22 wherein the blocking agent ofnon-specific binding is selected from the group consisting of gelatinand bovine serum albumin.
 24. The antigen diluent or buffer of claim 23wherein the blocking agent of non-specific binding is gelatin.
 25. Theantigen diluent or buffer of claim 24 wherein the concentration ofgelatin is from 0.05% to about 1.0%.
 26. The antigen diluent or bufferof claim 1 or 2 further comprising a chaotropic agent.
 27. The antigendiluent or buffer of claim 26 wherein the chaotropic agent is selectedfrom the group consisting of sodium thiocyanate and ammoniumthiocyanate.
 28. The antigen diluent or buffer of claim 27 wherein thechaotropic agent is ammonium thiocyanate
 29. The antigen diluent orbuffer of claim 28 wherein the concentration of ammonium thiocyanate isfrom about 10 mM to about 500 mM.
 30. The antigen diluent or buffer ofclaim 1 or 2 further comprising a buffering agent, a chelating agent, ablocking agent of non-specific binding, a chaotropic agent, anantibacterial agent, and a detergent.
 31. The antigen diluent or bufferof claim 30 wherein said buffering agent is sodium phosphate, saidchelating agent is EDTA, said blocking agent of non-specific binding isgelatin, said chaotropic agent is sodium thiocyanate, said antibacterialagent is sodium azide, and said detergent is SDS.
 32. The antigendiluent or buffer of claim 31 comprising 25 mM sodium phosphate, pH 6.5,5 mM EDTA, 10 mM DTT, 0.2% gelatin, 100 mM ammonium thiocyanate, 0.09%sodium azide and 0.1% SDS.
 33. The antigen diluent or buffer of claim 31comprising 50 mM sodium phosphate, 5 mM EDTA, 100 mM ammoniumthiocyanate, 0.06% SDS, 0.25% fish gelatin and 10 mM DTT.
 34. Animproved immunoassay kit for the detection of HCV antibodies wherein theimprovement comprises an antigen diluent or buffer for HCV antigenscomprising a reducing agent.
 35. The improved immunoassay kit of claim 6wherein the reducing agent is selected from the group consisting ofdithiothreitol, thioglycerol and mercaptoethanol.
 36. The improvedimmunoassay kit of claim 6 wherein the reducing agent is dithiothreitol.37. An improved assay for the detection of antibodies directed againsthepatitis C virus antigens wherein the improvement comprises the antigendiluent or buffer of claim 1 .
 38. A composition comprising at least oneantigen and an antigen diluent or buffer comprising a reducing agent.39. A composition comprising at least one HCV antigen and an antigendiluent or buffer comprising a reducing agent.